Cloning, purification and substrate conformations of aminoglycoside acetyltransferase (3)-IIIb

The aminoglycoside resistance protein aminoglycoside acetyltransferase (3)-IIIb (AAC3- nib) was successfully cloned, expressed and purified. This will allow many future experiments to be done using AAC3-IIIb. Using 2D NMR and molecular modeling, the conformations of two aminoglycosides, kanamycin and ribostamycin, were determined when bound to AAC3-lIIb. The structures determined for ribostamycin were divided into two conformers defined by the phi-psi angles of the glycosidic bonds as follows: Conformer 1 (11 total): Φ1A = -22 +/- 3, &Psi,1A= -42 +/-1, Φ1C= -9 +/- 4, Ψ1C = 51 +/-1 and for Conformer 2 (11 total): Φ1A = -67 +/- 0.7, Ψ1A = -59 +/- 0.8, Φ1C = -9 +/-3, Ψ1C = 49 +/-1. This gives the C to B ring orientation with respect to one another to be the same in both conformers. For kanamycin there were three conformers determined using the phi-psi angles as follows: Conformer 1 (9 structures): Φ1A = -11 +/- 3, Ψ1A= - 45 +/- 3, Φ1C= -9 +/- 4, Ψ1C = 50 +/- 2; Conformer 2 (6 structures): Φ1A = -8 +/- 3, Ψ1A = 48 +/- 2, Φ1C= -18 +/- 2, Ψ1C = -42 +/-2; and Conformer 3 (5 structures): Phi;1A = -73 +/- 0.6, Psi;1A= -57 +/- 0.2, Φ1C -14 +/- 0.5, Ψ1C = 51 +/- 0.2. Because the B to C orientation was the same for Conformer 1 and Conformer 3, this gives 14 out of 20 structures with that conformation for these rings. The C ring appears to be more constrained when either aminoglycoside is used while the A ring is somewhat more flexible. However, a comparison of the two aminoglycosides gives the A ring in one orientation (Conformer 1 for both ribostamycin and kanamycin) more than the others. This suggests that this may be the proper orientation of this ring. It has been suggested that the A and B rings provide the most important contributions when binding to both other aminoglycoside modifying enzymes and to RNA (Fourmy et al, 1996; Yoshizawa et al, 1998; Serpersu et al, 2000). Comparisons superimposing these rings with other enzyme bound aminoglycosides as well as RNA bound aminoglycosides shows remarkable similarity among the conformations. This gives important conformational information that may be used in future drug and enzyme inhibitor design studies

Diet-induced early-stage atherosclerosis in baboons: lipoproteins, atherogenesis, and arterial compliance

Background The purpose of this study was to determine if dietary manipulation can reliably induce early-stage atherosclerosis and clinically relevant changes in vascular function in an established, well-characterized nonhuman primate model. Methods We fed 112 baboons a high cholesterol, high fat challenge diet for two years. We assayed circulating biomarkers of cardiovascular disease (CVD) risk, at 0, 7, and 104 weeks into the challenge; assessed arterial compliance noninvasively at 104 weeks; and measured atherosclerotic lesions in three major arteries at necropsy. Results We observed evidence of atherosclerosis in all but one baboon fed the two-year challenge diet. CVD risk biomarkers, the prevalence, size, and complexity of arterial lesions, plus consequent arterial stiffness, were increased in comparison to dietary control animals. Conclusions Feeding baboons a high cholesterol, high fat diet for two years reliably induces atherosclerosis, with risk factor profiles, arterial lesions, and changes in vascular function also seen in humans

Histological variation of early stage atherosclerotic lesions in baboons after prolonged challenge with high-cholesterol, highfat diet

Introduction—The baboon is a well-characterized model of human early stage atherosclerosis. However, histological and morphological changes involved in atherogenesis in baboons are not known. Previously we challenged baboons with a high-cholesterol, high-fat diet for two years and observed fatty streak and plaque lesions in iliac arteries (RCIA). Methods—We evaluated histological and morphological changes of baboon arterial lesions and control arteries. In addition, we evaluated the vascular expression of CD68 and SMαA markers with progression of atherosclerosis. Results—We observed changes that correlated with extent of atherosclerosis, including increased maximum intimal thickness. We demonstrated at molecular level the infiltration of smooth muscle cells and macrophages into the intimal layer. Further, we observed histological and morphological discordancy between the affected and adjacent areas of the same RCIA. Conclusion—Atherogenesis in baboons is accompanied by histological, morphological and molecular changes, as in humans, providing insights to evaluate the mechanisms underlying early stage atherosclerosis in target tissues

Downhole Pumping

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General anaesthetic and airway management practice for obstetric surgery in England: a prospective, multi-centre observational study

There are no current descriptions of general anaesthesia characteristics for obstetric surgery, despite recent changes to patient baseline characteristics and airway management guidelines. This analysis of data from the direct reporting of awareness in maternity patients' (DREAMY) study of accidental awareness during obstetric anaesthesia aimed to describe practice for obstetric general anaesthesia in England and compare with earlier surveys and best-practice recommendations. Consenting patients who received general anaesthesia for obstetric surgery in 72 hospitals from May 2017 to August 2018 were included. Baseline characteristics, airway management, anaesthetic techniques and major complications were collected. Descriptive analysis, binary logistic regression modelling and comparisons with earlier data were conducted. Data were collected from 3117 procedures, including 2554 (81.9%) caesarean deliveries. Thiopental was the induction drug in 1649 (52.9%) patients, compared with propofol in 1419 (45.5%). Suxamethonium was the neuromuscular blocking drug for tracheal intubation in 2631 (86.1%), compared with rocuronium in 367 (11.8%). Difficult tracheal intubation was reported in 1 in 19 (95%CI 1 in 16-22) and failed intubation in 1 in 312 (95%CI 1 in 169-667). Obese patients were over-represented compared with national baselines and associated with difficult, but not failed intubation. There was more evidence of change in practice for induction drugs (increased use of propofol) than neuromuscular blocking drugs (suxamethonium remains the most popular). There was evidence of improvement in practice, with increased monitoring and reversal of neuromuscular blockade (although this remains suboptimal). Despite a high risk of difficult intubation in this population, videolaryngoscopy was rarely used (1.9%)

Identification of Inhibitors of Yellow Fever Virus Replication Using a Replicon-Based High-Throughput Assay▿

Flaviviruses cause severe disease in humans and are a public health priority worldwide. However, no effective therapies or drugs are commercially available yet. Several flavivirus replicon-based assays amenable to high-throughput screening of inhibitors have been reported recently. We developed and performed a replicon-based high-throughput assay for screening small-molecule inhibitors of yellow fever virus (YFV) replication. This assay utilized packaged pseudoinfectious particles containing a YFV replicon that expresses Renilla luciferase in a replication-dependent manner. Several small-molecule compounds with inhibitory activity at micromolar concentrations were identified in the high-throughput screen. These compounds were subsequently tested for their inhibitory activities against YFV replication and propagation in low-throughput assays. Furthermore, YFV mutants that escaped inhibition by two of the compounds were isolated, and in both cases, the mutations were mapped to the NS4B coding region, suggesting a novel inhibitory target for these compounds. This study opens up new avenues for pursuing the nonenzymatic nonstructural proteins as targets for antivirals against YFV and other flaviviruses

Analysis of Prostate-Specific Antigen Transcripts in Chimpanzees, Cynomolgus Monkeys, Baboons, and African Green Monkeys

<div><p>The function of prostate-specific antigen (PSA) is to liquefy the semen coagulum so that the released sperm can fuse with the ovum. Fifteen spliced variants of the <i>PSA</i> gene have been reported in humans, but little is known about alternative splicing in nonhuman primates. Positive selection has been reported in sex- and reproductive-related genes from sea urchins to <i>Drosophila</i> to humans; however, there are few studies of adaptive evolution of the <i>PSA</i> gene. Here, using polymerase chain reaction (PCR) product cloning and sequencing, we study <i>PSA</i> transcript variant heterogeneity in the prostates of chimpanzees (<i>Pan troglodytes</i>), cynomolgus monkeys (<i>Macaca fascicularis</i>), baboons <i>(Papio hamadryas anubis),</i> and African green monkeys <i>(Chlorocebus aethiops)</i>. Six <i>PSA</i> variants were identified in the chimpanzee prostate, but only two variants were found in cynomolgus monkeys, baboons, and African green monkeys. In the chimpanzee the full-length transcript is expressed at the same magnitude as the transcripts that retain intron 3. We have found previously unidentified splice variants of the <i>PSA</i> gene, some of which might be linked to disease conditions. Selection on the <i>PSA</i> gene was studied in 11 primate species by computational methods using the sequences reported here for African green monkey, cynomolgus monkey, baboon, and chimpanzee and other sequences available in public databases. A codon-based analysis (dN/dS) of the <i>PSA</i> gene identified potential adaptive evolution at five residue sites (Arg45, Lys70, Gln144, Pro189, and Thr203).</p></div